CORRELATION BETWEEN PHOSPHORYLATION OF THE CHEMOTAXIS PROTEIN-CHEY AND ITS ACTIVITY AT THE FLAGELLAR MOTOR
CORRELATION BETWEEN PHOSPHORYLATION OF THE CHEMOTAXIS PROTEIN-CHEY AND ITS ACTIVITY AT THE FLAGELLAR MOTOR
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DOI：
10.1021/bi00121a034
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发表时间：
1992-02-18
期刊：
影响因子：
2.9
通讯作者：
EISENBACH, M
中科院分区：
文献类型：
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作者：
BARAK, R;EISENBACH, M
趋化蛋白CheY被其激酶CheA磷酸化似乎在细菌趋化性的信号转导过程中起核心作用。据推测，该作用是CheY的激活，其导致鞭毛顺时针（CW）旋转。这项研究的目的是确定CheY的这种活性是否确实取决于被磷酸化的蛋白质。由于磷酸化的CheY只能在体外检测，我们研究了CheY的能力，导致CW旋转在体外系统中，由无细胞质的信封鼠伤寒沙门氏菌或大肠杆菌具有功能性鞭毛。仅包含缓冲区的封套仅逆时针旋转。包含CheY导致14%的旋转包络顺时针旋转。这部分的CW-旋转信封没有改变时，在信封中的磷酸电位降低，包括ADP与CheY在其中，表明CheY具有一定程度的活性，即使没有被磷酸化。试图通过磷酸化增加包膜中CheY的活性没有成功。然而，当CheY插入到部分裂解的细胞（半包膜）磷酸化条件下，CW旋转细胞的数量增加了3倍。这相当于磷酸化后单个CheY分子的活性增加了100倍以上。它的结论是，nonphosphorylated CheY可以相互作用的鞭毛开关，并导致CW旋转，但这种活动增加了至少2个数量级的磷酸化。这种活性的增加需要额外的细胞质成分，其身份尚不清楚。
Phosphorylation of the chemotaxis protein CheY by its kinase CheA appears to play a central role in the process of signal transduction in bacterial chemotaxis. It is presumed that the role is activation of CheY which results in clockwise (CW) flagellar rotation. The aim of this study was to determine whether this activity of CheY indeed depends on the protein being phosphorylated. Since the phosphorylation of CheY can be detected only in vitro, we studied the ability of CheY to cause CW rotation in an in vitro system, consisting of cytoplasm-free envelopes of Salmonella typhimurium or Escherichia coli having functional flagella. Envelopes containing just buffer rotated only counterclockwise. Inclusion of CheY caused 14% of the rotating envelopes to go CW. This fraction of CW-rotating envelopes was not altered when the phosphate potential in the envelopes was lowered by inclusion of ADP together with CheY in them, indicating that CheY has a certain degree of activity even without being phosphorylated. Attempts to increase the activity of CheY in the envelopes by phosphorylation were not successful. However, when CheY was inserted into partially-lysed cells (semienvelopes) under phosphorylating conditions, the number of CW-rotating cells increased 3-fold. This corresponds to more than a 100-fold increase in the activity of a single CheY molecule upon phosphorylation. It is concluded that nonphosphorylated CheY can interact with the flagellar switch and cause CW rotation, but that this activity is increased by at least 2 orders of magnitude by phosphorylation. This increase in activity requires additional cytoplasmic constituents, the identity of which is not yet known.